PROJECT SUMMARY In regenerative medicine, it is critically important to understand the complex mechanisms that rewrite and stably maintain cellular memory in order to reprogram cells to the new, desired destination fates. Wound healing, involving critical biological processes at multiple spatial and temporal scales, provides an ideal system for studying regenerative mechanisms. In skin, several distinct pools of epithelial stem cells, such as those in the interfollicular epidermis and different parts of the hair follicle, become activated and recruited to repair the wound. Importantly, large skin wounds can regenerate the normal array of tissue constituents, specifically new hairs, while small wounds never can. We hypothesize that regeneration is an emerging property arising from the optimal interplay between many biological events at multiple temporal and spatial scales including, but not limited to, transcriptional reprogramming of migrating epidermal, dermal and immune cells, as well as signaling crosstalk between these cells and their surrounding microenvironment.. Here, we propose a novel multiscale framework integrating multiple physiological systems (e.g. epidermal, dermal, and immune cells and hair follicles) to identify critical conditions for shifting injury repair toward regeneration and away from scarring. The proposed methodology addresses cutting-edge multiscale challenges in analyzing single-cell molecular data and their connections with spatial dynamics in tissues. We will carry out three aims. In Aim 1, we will identify regeneration-specific gene profile changes in epidermal, dermal, and immune cell in healing wounds; in Aim 2, we will develop an integrative multiscale model to predict the relative roles and emergent dynamics of multiple interacting cell types during wound healing; and in Aim 3, we will test model predictions using in-vivo murine functional assays and ex vivo human co-culture; in combination with multiscale simulations and statistical inference, we will thus be able to dissect the regenerative roles and spatial dynamics of candidate regulators. The knowledge gained in this proposed work will help to develop future protocols for augmenting the regeneration mechanisms in clinical settings to achieve robust human skin regeneration after any injury (small or large) and with high efficiency (i.e. always achieve high density of regenerating hairs). The overall insights learned will not only shed new light into skin research, but also establish a founding paradigm for other epithelial systems. The novel computational tools for single-cell RNA-seq-driven cell lineage tracking, the robust multiscale models for spatial dynamics of multiple cell lineages, and the overall integrative multiscale framework of tissue regeneration will have broad applications, including for embryonic development, solid tumors, and many other epithelial and even non-epithelial tissues. Given the importance of stem/progenitor cells in regeneration and tumorigenesis, these studies will also have important implications for tissue engineering and cancer treatment.